Bruce S. Seal NIH Public Access Poultry Microbiology...

Clostridium perfringens bacteriophages ΦCP39O and ΦCP26F:genomic organization and proteomic analysis of the virions

Bruce S. Seal,Poultry Microbiology Safety Research Unit, Richard B. Russell Agricultural Research Center,Agricultural Research Service, USDA, 950 College Station Road, Athens, GA 30605, USA

Derrick E. Fouts,J. Craig Venter Institute, 9712 Medical Center Drive, Rockville, MD 20850, USA

Mustafa Simmons,Poultry Microbiology Safety Research Unit, Richard B. Russell Agricultural Research Center,Agricultural Research Service, USDA, 950 College Station Road, Athens, GA 30605, USA

Johnna K. Garrish,Poultry Microbiology Safety Research Unit, Richard B. Russell Agricultural Research Center,Agricultural Research Service, USDA, 950 College Station Road, Athens, GA 30605, USA

Robin L. Kuntz,Poultry Microbiology Safety Research Unit, Richard B. Russell Agricultural Research Center,Agricultural Research Service, USDA, 950 College Station Road, Athens, GA 30605, USA

Rebekah Woolsey,Nevada Proteomics Center, University of Nevada, Reno/MS200, 1664 North Virginia Street,Reno, NV 89557, USA

Kathleen M. Schegg,Nevada Proteomics Center, University of Nevada, Reno/MS200, 1664 North Virginia Street,Reno, NV 89557, USA

Andrew M. Kropinski,Laboratory for Foodborne Diseases, Public Health Agency of Canada, Guelph, ON N1G 3W4,Canada. Molecular and Cellular Biology, University of Guelph, Guelph, ON N1G 3W4, Canada

Hans-W. Ackermann, andFelix d’Herelle Reference Center for Bacterial Viruses, Department of Medical Biology, Faculty ofMedicine, Laval University, Laval, QC G1K 7P4, Canada

Gregory R. Siragusa

© Springer-Verlag (outside the USA) 2010

Correspondence to: Bruce S. Seal, [email protected].

Present Address: G. R. Siragusa, Danisco USA, W227 N752 Westmound Dr., Waukesha, WI 53186, [email protected]

Electronic supplementary material The online version of this article (doi:10.1007/s00705-010-0812-z) contains supplementarymaterial, which is available to authorized users.

NIH Public AccessAuthor ManuscriptArch Virol. Author manuscript; available in PMC 2014 August 10.

Published in final edited form as:Arch Virol. 2011 January ; 156(1): 25–35. doi:10.1007/s00705-010-0812-z.

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Poultry Microbiology Safety Research Unit, Richard B. Russell Agricultural Research Center,Agricultural Research Service, USDA, 950 College Station Road, Athens, GA 30605, USA

Bruce S. Seal: [email protected]

Abstract

Poultry intestinal material, sewage and poultry processing drainage water were screened for

virulent Clostridium perfringens bacteriophages. Viruses isolated from broiler chicken offal

washes (O) and poultry feces (F), designated ΦCP39O and ΦCP26F, respectively, produced clear

plaques on host strains. Both bacteriophages had isometric heads of 57 nm in diameter with 100-

nm non-contractile tails characteristic of members of the family Siphoviridae in the order

Caudovirales. The double-strand DNA genome of bacteriophage ΦCP39O was 38,753 base pairs

(bp), while the ΦCP26F genome was 39,188 bp, with an average GC content of 30.3%. Both viral

genomes contained 62 potential open reading frames (ORFs) predicted to be encoded on one

strand. Among the ORFs, 29 predicted proteins had no known similarity while others encoded

putative bacteriophage capsid components such as a pre-neck/appendage, tail, tape measure and

portal proteins. Other genes encoded a predicted DNA primase, single-strand DNA-binding

protein, terminase, thymidylate synthase and a transcription factor. Potential lytic enzymes such as

a fibronectin-binding autolysin, an amidase/hydrolase and a holin were encoded in the viral

genomes. Several ORFs encoded proteins that gave BLASTP matches with proteins from

Clostridium spp. and other Gram-positive bacterial and bacteriophage genomes as well as

unknown putative Collinsella aerofaciens proteins. Proteomics analysis of the purified viruses

resulted in the identification of the putative pre-neck/appendage protein and a minor structural

protein encoded by large open reading frames. Variants of the portal protein were identified, and

several mycobacteriophage gp6-like protein variants were detected in large amounts relative to

other virion proteins. The predicted amino acid sequences of the pre-neck/appendage proteins had

major differences in the central portion of the protein between the two phage gene products. Based

on phylogenetic analysis of the large terminase protein, these phages are predicted to be pac-type,

using a head-full DNA packaging strategy.

Introduction

Clostridium perfringens is a Gram-positive, spore-forming, anaerobic bacterium that is

commonly present in the intestines of humans and animals. C. perfringens strains are

classified into five types (A, B, C, D, or E) based on the toxin they produce [69, 75]. Spores

of the pathogen can persist in soil, feces or the environment, and the bacterium causes many

severe infections of animals and humans. The bacterium can cause food poisoning, gas

gangrene (clostridial myonecrosis), enteritis necroticans, and non-foodborne gastrointestinal

infections in humans and is also a veterinary pathogen, causing enteric diseases in both

domestic and wild animals [69, 75]. This organism is considered the cause of necrotic

enteritis in chickens, and although this does not necessarily present a threat of human illness,

it could potentially become a far greater problem for the poultry industry if antibiotics are

withdrawn from animal feeds, as is the case in the European Union [81].

Although there are now sequences of C. perfringens genomes available [60, 73], there is a

paucity of data for C. perfringens bacteriophages. Temperate and virulent phages are

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associated with the bacterium, for which there is no genomic sequence [12, 19, 37, 40, 50,

51, 62, 65, 76, 79, 82], and a phage-typing system was developed for the organism [86].

Zimmer et al. [88] isolated two temperate phages by UV irradiation (Φ3626 and Φ8533) of

lysogenic C. perfringens cultures. The genome of phage Φ3626 was determined to be 33.5

kb, containing 50 potential open reading frames (ORF) with 3′-protruding cohesive ends.

Only 19 of these gene products could be assigned to potential biological functions based on

bioinformatics analysis. Several of these potentially influence cell spore formation due to the

presence of phage genes in the bacterial genome. Subsequently, these investigators also

identified a phage-specific enzyme identified as a murein hydrolase. The lysin had lytic

activity against 48 test cultures of C. Perfringens but was not active against members of

other clostridial species or bacteria belonging to other genera [89].

There has been a resurgent interest in bacteriophage biology and the use of phages or their

gene products as antibacterial agents [28, 45, 57]. The potential application of

bacteriophages and/or their lytic enzymes has been of considerable interest for human and

veterinary medicine as well as the bioindustry worldwide due to antibiotic resistance of

human bacterial pathogens [47, 81]. The importance of phages to bacterial evolution [2, 15],

the role of phage- or prophage-encoded virulence factors that contribute to bacterial

infectious diseases [11] and the contribution of phages to horizontal gene transfer [15]

cannot be overstated. Additionally, their contribution to microbial ecology [64] and

agricultural production [16, 80] is also extremely important. Bacteriophages play important

roles in related clostridial species that include toxin gene transfer and toxin production, and

they may even affect physiological functions of the host bacterium such as sporulation [13,

63]. Although the role of phage in the pathogenesis of C. botulinum [25, 26] and, more

recently, C. difficile [32, 33, 36] has been documented, this is not the case for C.

perfringens.

Clostridium perfringens plays important roles in human food-borne disease and causes

diseases among poultry or other animals [69] that are of concern for human health [81]. We

are identifying new antimicrobial agents, such as putative lytic enzymes from the genomes

of bacteriophages, that are active against C. perfringens. Consequently, bacterial viruses

capable of lysing strains of C. perfringens were initially characterized by negative staining

and transmission electron microscopy. Two clear-plaque bacteriophage isolates designated

ΦCP39O and ΦCP26F were chosen for genomic and proteomic analysis.

Materials and methods

Bacterial hosts, bacteriophage isolation and propagation

Clostridium perfringens isolates 26 and 39, utilized as hosts for propagation of

bacteriophages, were cultured in brain heart infusion (BHI) broth or on agar (Remel,

Lenexa, KS) and characterized by 16S rRNA-DNA sequence analysis as described

previously [23, 74, 85]. Offal washes (O) and feces (F) obtained at a local chicken-

processing facility were clarified by low-speed centrifugation (5,000×g for 20 min at 5°C),

followed by filtration of the supernatant, first through cheesecloth, and then through 0.45-

μm bottle filters (Corning Inc., Corning, NY). Bacterial viruses producing clear plaques on

C. perfringens were identified by spot-testing and titration on strains [76, 88] susceptible to

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the isolated phages. Several clostridial species including C. absonum, C. acetobutylicum, C.

beijerinckii, C. novyi, C. rubrum, C. sordellii, C. sporogenes, C. tetani, and C.

tetanomorphum [74, 85] were spot-tested for lytic activity [76, 88]. The C. perfringens-

specific bacteriophages ΦCP39O and ΦCP26F were propagated by plating with low-melt

agar using the C. perfringens isolates 39 and 26 cultured at 37°C in the Anaero

Pack™(Mitsubishi Gas Chemical Co., Japan) system with AnaeroGen (OXOID Ltd.,

Basingstoke, England) sachets [76, 88]. The bacteriophages were subjected to three rounds

of plaque purification and consistently suspended in TMGS (10 mM Tris, pH 8, 10 mM Mg

++, 0.55% NaCl, 0.1% gelatin) at an average titer of 2 × 108 pfu/ml. Subsequently,

bacteriophages ΦCP39O and ΦCP26F were propagated utilizing a plate lysis method [38]

under anaerobic conditions for virus purification and DNA extraction.

Purification of bacteriophages, genomic DNA purification and electron microscopy

After plate lysis [38] in anaerobic chambers, bacteriophage genomic DNA was purified

using the Qiagen Lambda Phage DNA isolation protocol (Qiagen, Valencia, CA, USA).

Additionally, bacteriophages were purified from plate lysates by centrifugation at 2,000×g

for 20 min to remove bacterial debris and low-melt agarose. The clarified supernatant was

centrifuged at 103,800×g for 90 min, followed by suspension of the phage pellet in 1 ml

TBS (20 mM Tris and 500 mM NaCl at pH 7.5). The bacteriophage suspensions were

layered over a 15–55% sucrose-TB gradient and centrifuged at 103,800×g in a Beckman JS

24.38 rotor for 90 min [58] and further purified through 20–50% continuous potassium

tartrate density gradients [31, 55, 58] by centrifugtion at 105,000×g in a Beckman JS 24.15

rotor for 90 min. The bacteriophage bands were drawn from the gradient, diluted in TBS and

concentrated by centrifugation at 105,000×g in a Beckman JS 24.15 rotor for 90 min,

followed by suspension in TBS. Bacteriophages were stained with phosphotungstate (270,

pH 7.2) and examined in a Philips EM 300 electron microscope. Magnification was

controlled by means of T4 phage tails [3]. The bacteriophage pellets were also subjected to

proteinase K (20 μg/ml) digestion in the presence of 0.1% sarcosyl and 0.2 M EDTA,

followed by phenol–chloroform extraction and ethanol precipitation to obtain genomic DNA

[68].

Molecular cloning, sequencing, annotation of genomic DNA and phylogenetic analysis

After purification of bacteriophage genomic DNA, the nucleic acid was subjected to

spectrophotometer readings at 260/280 nm and restriction enzyme digestion, followed by

agarose gel electrophoresis [67]. Sequencing of the bacteriophage genomes was completed

by MWG Biotech, (High Point, NC, USA), by the J. Craig Venter Institute [29], and

pyrosequencing [53] was done at the National Microbiology Laboratory (Winnipeg, MB).

For Sanger sequencing, phage DNA was sheared using a nebulizer, blunt-end repaired and

dephosphorylated [68]. DNA fragments of the desired size (1 to 4 kb) were ligated into

pSmart (Lucigen™) for propagation in E. coli after transformation. Clones were sequenced

such that approximately 14-fold redundancy was obtained for the genome including primer

walking to fill gaps [29]. To check accuracy, phage DNA was cleaved with the restriction

enzymes HindIII, EcoRI, EcoRV, AluI and ClaI (New England Biolabs, Ipswich, MA),

treated with Taq polymerase [44] and cloned [56] into the TOPO TA vector (Invitrogen,

Carlsbad, CA). Additionally, end-repair and G-tailing were used for cloning restriction

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enzyme fragments into pSmart vectors (Lucigen, Middleton, WI) for nucleotide sequencing

[77; Applied Biosystems Inc., Foster City, CA].

Nucleotide sequence assembly, editing, analysis, prediction of amino acid sequences and

alignments were conducted using the Celera Assembler [59], MacVector 7.2™ (Accelrys,

San Diego, CA) and DNASTAR™ (Madison, WI) software. Open reading frames (ORFs)

were predicted using GeneMark.hmm for prokaryotes [http://opal.biology.gatech.edu/

GeneMark; 49] and ORF Finder [http://ncbi.nlm.nih.gov/gorf/gorf.html] software. The

predicted protein amino acid sequences were searched using BLAST [6] and PSI-BLAST or

BLASTP [7, 70] as well as the conserved domain database [52] algorithms. The large

terminase protein was utilized to predict the mechanism of DNA packaging and the structure

of the genome ends [21, 29]. We inferred the tree with a J. Craig Venter Institute (JCVI) in-

house wrapper script around the PAUP* program [84]. The wrapper converted a FASTA-

format alignment created by MUSCLE [24] into the NEXUS format expected by PAUP*. In

addition, as trees bootstrapped by PAUP lose evolutionarily meaningful branch lengths (see

http://paup.csit.fsu.edu/paupfaq/faq.html), retaining only the bootstrap values themselves,

the wrapper fed the PAUP-generated tree along with the alignment into TREE-PUZZLE

[71] to infer the maximum-likelihood branch length values for the tree.

Preparation of purified virions and two-dimensional (2D) gel electrophoresis

Bacteriophage protein purification was done by adding four volumes of acetone at −20°C to

the centrifuged phage pellet for at least 1 h. After centrifugation at 16,000×g for 10 min at

4°C, the pellet was washed three times in cold acetone/water (4:1), followed by

centrifugation, and dried under vacuum [22]. To ensure reproducible gel electrophoresis,

purified virion protein fractions were digested with N-glycosidase F (PNGase F) to cleave

any potential oligosaccharides prior to extraction with acetone as per the manufacturer’s

instructions (New England Biolabs, Ipswich, MA) for 1 h at 37°C. The water–acetone

pellets were further purified following the procedure for “2-D gel clean-up” to remove

interfering substances such as salts, detergents, lipids and phenolics (Amersham

Biosciences, Piscataway, NJ).

Proteomics analysis was carried out using 2-D gel electrophoresis [22, 61]. Protein samples

were suspended in electrophoresis buffer (5 M urea, 2 M thiourea, 2% CHAPS, 2% SB3-10,

0.2% 3/10 ampholyte with 40 mM Tris, pH 7.4), mixed by vortexing and centrifuged at

16,000×g at 22°C for 10 min. Supernatant proteins suspended in electrophoresis buffer were

separated by isoelectric focusing (pH 3–10) in the first dimension, followed by SDS–PAGE

and staining with Coomassie blue [22, 61]. Spot detection and pattern matching were done

qualitatively on a Bio-Rad VersaDoc 4000 imager, analyzing gels from each isolate in

triplicate with PDQuest version 7.3.0 (Bio-Rad, Hercules, CA). Protein spots were cut using

a BioRad EXQuest spot cutter and digested using Genomic Solutions Proprep [30].

Identification of purified bacteriophage proteins by mass spectrometry

Tryptic peptide molecular masses obtained by MALDI-TOF-TOF MS [4, 42] were utilized

to identify proteins by searching the protein sequence Mascot database at the National

Center for Biotechnology Information, PIR and Swiss-Prot with ProFound (Proteomics,

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http://opal.biology.gatech.edu/GeneMark

http://opal.biology.gatech.edu/GeneMark

http://ncbi.nlm.nih.gov/gorf/gorf.html

http://paup.csit.fsu.edu/paupfaq/faq.html

New York, NY) and the predicted amino acid sequences from the bacteriophage genomes.

Specifically, samples were prepared and spotted onto a MALDI (matrix-assisted laser

desorption/ionization) target using ZipTipu-C18 from Millipore (Billerica, MA). Samples

were aspirated and dispensed with ZipTipu-C18 and eluted with conditioning solution (70%

acetonitrile, 0.2% formic acid) containing 5 mg/ml MALDI matrix (α-cyano-4-

hydroxycinnamic acid), and 0.5 μl was spotted onto the MALDI target.

Samples were analyzed using a model 4700 Proteomics Analyzer with TOF/TOF Optics

(Applied Biosystems, Foster City, CA). MALDI-MS data were collected in the m/z range of

700 to 4,000 using trypsin autolysis products with m/z ratios of 842.51 and 2211.10 as

internal standards. Data were analyzed using GPS Explorer Software (Applied Biosystems).

The data were acquired in reflector mode from a mass range of 700–4,000 Da, and 1,250

laser shots were averaged for each mass spectrum. Each sample was internally calibrated if

both the 842.51 and 2211.10 ions from trypsin autolysis were present. The eight most

intense ions from the MS analysis that were not on the exclusion list were subjected to

MS/MS. For MS/MS analysis, the mass range was 70 to precursor ion with a precursor

window of −1 to 3 Da, with an average 5,000 laser shots for each spectrum.

The peptide data were extracted from the Oracle database, and a peak list was created by

GPS Explorer software (Applied Biosystems Inc.,) from the raw data generated from the

ABI 4700. This peak list was based on signal-to-noise filtering and an exclusion list and

included de-isotoping. The resulting file was then searched by Mascot (Matrix Science,

Boston, MA). A tolerance of 20 ppm was used if the sample was internally calibrated, and

200 ppm tolerance was used if the default calibration was applied. Protein identification was

validated by the following criteria:>20 ppm mass accuracy for all MS ions, and all ions,

which were not modified, had to be accounted for in at least two MS/MS spectra. Database

search parameters included one missed cleavage, oxidation of methionines and

carbamidomethylation of cysteines.

Results and discussion

Isolation of bacteriophages that are virulent for Clostridium perfringens

Bacteriophages ΦCP39O and ΦCP26F, which are capable of lysing C. perfringens isolates

39 and 26, were identified by limit dilution, followed by spot-testing and titration on the host

with 0.2-μm-filtered chicken offal wash (O) or feces (F). Both the of viruses produced clear

plaques after limit dilution plating of the virus on their respective hosts (Fig. 1a), and both

viruses were restricted in range to their specific hosts. Also, only clear plaques were

detected upon repeated propagation, with no indication of lysogeny. Rather than CsCl

density gradient centrifugation for purification, the bacteriophages were purified by

potassium tartrate gradient centrifugation [31], which produced pure phage preparations

with morphologically intact particles containing nucleic acid. Both bacteriophages had

equivalent morphologies that were characteristic of members of the family Siphoviridae,

order Caudovirales [1, 2], and had isometric heads of 57 nm between apices of rigid 100-

by-7 nm non-contractile tails with terminal knobs (Fig. 1b). ΦCP39O and ΦCP26F differed

in their dimensions from other known C. perfringens phages, most of which were members

of the family Podoviridae that had short non-contractile tails with a collar and collar fibers

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[12, 62, 76]. The bacteriophages resemble some C. perfringens siphoviruses [51, 88] but

differ from others by their considerably shorter tails [37, 40].

Nucleotide sequences of bacteriophage genomes and predicted open reading frames

The double-strand (ds) DNA genome of bacteriophage ΦCP39O was 38,753 base pairs (bp)

long with a 30.4% GC content. It contained 62 predicted open reading frames (ORFs). The

ds DNA genome of bacteriophage ΦCP26F was 39,188 bp with a 30.3% GC content and

also contained 62 predicted ORFs. The genomes of ΦCP39O and ΦCP26F shared 96.6%

identity, and the genome maps were arranged as a linear model (Fig. 2) encoding proteins

predicted or similar to putative proteins found in the ORFs of other bacteriophages or

bacterial genomes (Tables S1 and S2, as online resources 1 and 2, respectively). However,

29 ORFs encode putative proteins that had no known similarity to others in the databases.

All but one ORF were predicted to be transcribed from one strand, and two genes, CDS05 of

ΦCP26F (Fig. 2; Table S2) and CDS12 of ΦCP39O (Fig. 2; Table S1), were present and

absent, respectively, in the bacteriophage genomes. The genomes of ΦCP39O and ΦCP26F

were similar in size to that of the previously reported C. perfringens Φ3626 [88], but smaller

than those of bacteriophages isolated from C. difficile [32, 33, 36, 54]. Although the genome

organization of these bacteriophages had a modular arrangement similar to that of many

phages [20], the overall sequence similarity was comparatively low, being only 46% with

the ΦSM101 genome [NC_008265; 60] and less than 20% similar to the only other

published C. perfringens phage genome, Φ3626 [NC_003524; 88]. Also, no putative genes

encoding clostridial toxin were identified in the genomes of ΦCP39O and ΦCP26F.

The genomes of both bacteriophages contained genes (Tables S1 and S2) encoding several

predicted proteins involved in DNA packaging and morphogenesis, including a putative

small terminase (CDS01) and a predicted terminase large subunit (CDS02) that was

homologous to a protein detected in the C. thermocellum genome [YP_001038869]. The

putative large terminase identified in the ΦCP39O and ΦCP26F genomes was most similar

to those of Staphylococcus spp. phages [41]. A gene encoding the putative bacteriophage

portal protein (CDS04) for DNA packaging and injection was also present in clostridial

genomes [10, 72] other than C. perfringens. Reportedly, the two most conserved tailed-

bacteriophage proteins are those involved in DNA packaging, the large terminase and portal

proteins [20]. In a BLAST search of ΦCP39O and ΦCP26F, the predicted portal proteins

(from CDS04) were most closely related to a protein found in Clostridium difficile

bacteriophages ΦCD119 [35] and ΦC2 [33], Bacillus subtilis phage ΦSPP1 [5] and

staphylococcal phages [41].

Putative minor head structural proteins from CDS05 of ΦCP39O and CDS06 of ΦCP26F

were similar to Bacillus subtilis phage ΦSPP1gp7, which is required for viral head

morphogenesis [8]. A Staphylococcus phage ΦROSA ORF19-like protein [41] was also

encoded in the genomes (CDS07 of ΦCP39O; CDS08 of ΦCP26F). The putative scaffold

protein (CDS13), which is presumably involved in conformational changes to ensure

efficiency and fidelity of assembly [27], and a pre-neck/appendage-like protein (CDS15)

were most similar to putative proteins encoded in the genomes of C. novyi [10] and C. tetani

[14] as well as to those reported in Staphylococcus phages [41]. A conserved hypothetical

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protein (CDS14) of ΦCP39O and ΦCP26F that was also predicted in C. perfringens

genomes [ZP_02864210] was most similar to the gp6 protein of several mycobacteriophages

[NP_817344; 66] and another phage protein that has been reported to be the capsid protein

of a freshwater cyanophage [YP_001285797; 46]. The putative bacteriophage tail protein

(CDS24) encoded in the ΦCP39O and ΦCP26F genomes was similar to that reported for the

C. perfringens Φ3626 gp14 [88]. The ΦCP39O and ΦCP26F tail proteins were also similar

to the C. difficile ΦCD119 ORF17 product [35] and the C. difficile ΦCD27 gp16 [54]. The

predicted tape-measure proteins (CDS22) of ΦCP39O and ΦCP26F were similar to the

ORF13 gene product of C. perfringens Φ3626, which was also reported to be a tape-measure

protein [88].

Several ORFs encoded proteins that are potentially involved in nucleic acid replication or

regulation (Tables S1 and S2). One of the predicted phage anti-repressor/transcription factor

proteins encoded by CDS23 was similar to a predicted ORF product in C. difficle [72].

Another potential phage anti-repressor protein gene (CDS32) was located adjacent to a

predicted DNA-binding protein gene (CDS31) that was also reported in Streptococcus phage

ΦSfi21 [48] and Staphylococcus spp. phage Φ71 [41]. A putative phage anti-repressor of

ΦCP39O and ΦCP26F (CDS43) was similar to a predicted C. difficile phage ΦC2 anti-

repressor protein [33]. Also present in the ΦCP39O and ΦCP26F genomes were a single-

strand DNA-binding protein gene (CDS52) that was also identified in the C. perfringens

genome [60] and a recombination protein gene (CDS48) that is distantly related to the

bacteriophage Φ42e DNA-binding protein gene [41]. A putative flavin-dependent

thymidylate synthase gene that has been reported in the C. perfringens genome [60] was also

encoded in the ΦCP39O genome (CDS51). The gene product of CDS54 was a predicted

XRE-family-like transcriptional regulator protein similar to elements identified in

Streptococcus thermophilus with possible integrative and conjugative functions [17], and its

gene was located adjacent to an ORF (CDS55) encoding a potential DNA primase Zn-finger

domain.

Two ORFs located contiguously on the ΦCP39O and ΦCP26F genomes (Tables S1 and S2)

encoded a predicted N-acetylmuramoyl-L-alanine amidase/hydrolase (CDS27) and the

putative phage holin (CDS28). These proteins are required for host-cell lysis and release of

phage progeny during late infection [9, 47]. The putative amidase of phage ΦCP39O was

predicted to be a 213-amino-acid protein, while the putative amidase of phage ΦCP26F was

a 212-residue protein presumably involved in cleaving the amide bond between N-

acetylmuramoyl and L-amino acids in bacterial cell walls or cell envelope biogenesis of the

outer membrane [78]. The predicted holin protein gene encoded a 75-aminoacid molecule

that had sequence similarity to hypothetical proteins found in C. perfringens [YP_695712;

NP_561927], Listeria phage ΦB054 [YP_001468736] and Streptococcus bacteriophages

[NP_049941; NP_056698]. The predicted phage ΦCP39O and ΦCP26F holin proteins do

not have the M-L-M motif of class I or II holins, so it is most likely a class III holin with a

single transmembrane domain [87], since the protein was predicted to primarily be

hydrophobic with low surface probability in the central region of the molecule, while the N-

and C-terminal ends were predicted to be hydrophilic.

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Proteomics analysis of purified bacteriophage ΦCP39O

Following 2D-gel electrophoresis of the purified virion (Fig. 3), the proteins were analyzed

by mass spectrometry (Table S3 as online resource 3). Four principal virion protein regions

were identified, including a portal protein (CDS04), a conserved hypothetical clostridial

protein (CDS14) that was determined to be mycobacteriophage gp6-like, a pre-neck

appendage protein (CDS15) and a minor structural protein (CDS25). The predicted portal

protein region corresponding to CDS04 was identified as a protein of approximately 50 kDa

(Fig. 3). The predicted size of the conserved hypothetical mycobacteriophage gp6-like

protein from CDS14 was approximately 30 kDa (Table S1), and this is consistent with its

relative mobility in the gel (Fig. 3). This protein is also homologous to the pfam11651 coat

protein of gene product 5 from bacteriophage P22, which is involved in the formation of

procapsid shells [43]. There are 415 molecules of the bacteriophage P22 coat protein

arranged in an icosahedral morphology [43], and the CDS14 gene product occurred in the

greatest abundance. Consequently, this protein is probably the major capsid protein of the

bacteriophage. The proteins included several variants with differences in isoelectric point,

possibly due to posttranslational modification [34], since they were predicted to be highly

myristilated and phosphorylated at many residues.

A predicted minor structural protein with an approximate size of greater that 110 kDa was

identified. This minor structural protein, which is a product of CDS25 (Table S1), shares

approximately 30% sequence similarity to ORF15 of C. perfringens phage Φ3626, which

encodes a putative endopeptidase [88]. Interestingly, the minor structural protein also had

similarity in the C-terminal portion of the molecule to a putative choline-binding protein

with potential hydrolase activity found in the genome of C. perfringens [NP_562049]. These

bacterial enzymes have an N-terminal catalytic module and a C-terminal choline-binding

module (CBM) that attaches them to the bacterial surface [39].

The pre-neck/appendage protein, a product of CDS15 (Table S1), was identified at an

approximate relative mobility of 100 kDa (Fig. 3). The pre-neck/appendage proteins of

ΦCP39O and ΦCP26F had predicted amino acid sequences with an overall identity of 79%

between them. However, the proteins shared 100% sequence identity from residues one

through 170 and in the C-terminal portion of the protein beyond residue 828 of the

alignment. The major sequence differences occurred in the central portion of the protein

(Fig. S1 as online resource 4). The pre-neck/appendage-like protein is most similar to

predicted sequences encoded by C. novyi [YP_878235] and C. tetani [NP_782154]

genomes, with a region from residues 79 through 237 that has similarity to the bacterial

TolA proteins with a myosin-like domain involved in the translocation of group A colicins

[67]. The TolA protein is important to filamentous phages and colicins during the process of

import into E. coli [83], and consequently, the CDS15 gene product could also potentially be

involved in entry of phage ΦCP39O and ΦCP26F into their hosts.

Several proteins with no identifiable homologous function were identified as part of the

virion or co-purified with the virus. These corresponded to products of CDS16 and CDS17

with an approximate relative mobility of 10–15 kDa and the gene product of CDS36, with

an approximate relative mobility of less than 10 kDa and an extremely high pI of >9 (Fig. 3;

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Table S1). The proteins encoded by CDS16 and CDS17 had low identity (30%) to proteins

predicted in the Collinsella aerofaciens genome [ZP_01771283; ZP_01771282]. Another

hypothetical protein with no known homologous function was identified, with a relative

mobility of approximately 20 kDa, corresponding to a protein of CDS19 (Fig. 3; Table S1).

Phylogenetic analysis of the predicted terminase protein

Following BLAST analysis of the predicted phage proteins, the large terminase was

determined to be encoded by CDS02 of the ΦCP39O and ΦCP26F phage genomes (Tables

S1 and S2). Specifically, the predicted 50-kDa protein was most closely related to terminase

proteins encoded by putative prophage sequences identified in the genomes of various

clostridial species such as C. thermocellum [YP_001038869], C. botulinum

[YP_001254847; YP_001254222] and less closely related to C. kluyveri [YP_001396654] or

C. difficile [ZP_01804380]. The terminase was not closely related to the currently published

clostridial phage terminase proteins, but it was closer in sequence similarity to the prophage

ΦLambd-aCh01 terminase large subunit reported from Carboxydo-thermus

hydrogenoformans [YP_360500]. The most closely related bacteriophage terminase protein

was from Staphylococcus spp. phage Φ27 [YP_240074] reported by Kwan et al. [41].

Phylogenetic analysis of the available bacteriophage terminase proteins (Fig. 4; Table S4 as

online resource 5) demonstrated that the ΦCP39O and ΦCP26F phage termin-ases were

most closely related to ΦCh01, ΦCB01, ΦCB02, ΦCN01 and bacteriophage ΦSPP1 (Fig. 4).

This indicates that our clostridial phages are potentially pac-type bacteriophages, which use

a headfull genomic DNA packaging strategy [21].

The phage ΦCP39O and ΦCP26F portal proteins were most similar in sequence to the

bacteriophage ΦSPP1 portal protein, which, along with the terminase, functions to package

its genomic DNA by a head-full packaging mechanism [18]. Also, the principal virion

protein (CDS04) is similar to a coat protein of ΦP22 that undergoes head-full DNA

packaging [43]. This correlates well with the phylogenetic prediction for phages ΦCP39O

and ΦCP26F being pac-type bacteriophages.

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

Acknowledgments

The authors thank Mary Ard at the University of Georgia, College of Veterinary Medicine, for preliminary electronmicroscopy. The investigations were supported by the Agricultural Research Service, USDA CRIS project#6612-32000-046 (BSS, GRS, MS) and a non-funded CRADA #58-6612-7-175 with DEF at JCVI. The proteomicsportion of the project was supported by NIH Grant Number P20 RR-016464 from the INBRE Program of theNational Center for Research Resources (RW and KS). Sequencing was also supported by a Discovery Grant fromthe Natural Sciences and Engineering Research Council of Canada (AK). The genome sequences were submitted toGenBank with accession numbers EU588980 for the ΦCP39O genome and GQ443085 for the ΦCP26F genome.

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Fig. 1.Bacteriophage ΦCP39O and ΦCP26F plaques and electron microscopy of the purified

virions. a Plaques produced on the host strain Clostridium perfringens strain 39. bMorphology of bacteriophage ΦCP39O following purification by density gradient

purification. The bar represents 100 nm

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Fig. 2.Physical map of the bacteriophage ΦCP39O and ΦCP26F genomes. The linear map is based

on nucleotide sequences of the phage genomes and predicted open reading frames. Forward

arrows represent directional transcription from what would be considered the plus strand,

while the single reverse arrow represents possible transcription on the opposite strand.

Colored boxes indicate functional gene groups, while transcriptional terminators are

indicated by hairpin-type structures

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Fig. 3.Two-dimensional gel electrophoresis of purified bacteriophage ΦCP39O virion proteins.

After velocity and density gradient centrifugation, proteins of the purified virions were

subjected to isoelectric focusing in the first dimension at pI 4–10, followed by vertical SDS–

PAGE (8–12%) in the second dimension. The genome sequences from which the proteins

were encoded (CDS) are also noted in the figure. Results of the mass spectrometry analyses

can be found in supplemental Table S3

Seal et al.


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Fig. 4.Phylogenetic analysis of amino acid sequences of the large terminase of bacteriophage

ΦCP39O compared with other bacteriophage terminase proteins. Accession numbers for the

large terminase proteins can be found in the online supplemental information, Table S4

Seal et al.


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